**3.** *Salmonella*

The amounts of antimicrobials utilised in livestock are vast and often exceed those in humans. Data suggest that in the EU approximately 70% of antimicrobials were sold for use in livestock in 2014 [3]. The consumption of antimicrobials in humans and animals has indeed been associated with the occurrence of antimicrobial resistance (AMR) in zoonotic bacteria [3], which are the causative agents of zoonoses and can be transmitted directly between animals and humans or *via* the food chain. AMR in zoonotic bacteria is a subject of major concern.

Even though AMR is an ancient and naturally occurring phenomenon in some bacteria [4], the excessive use of antimicrobials in humans and livestock, as well as poor hygiene conditions and practices in the food production chain, accelerates the emergence of resistance in zoonotic bacteria [5]. The alarming consequence of AMR coupled with the paucity of novel antimicrobials is the rise in the frequency of multidrug resistant (MDR) zoonotic bacteria that may lead to an impaired response to antimicrobial therapy or ultimately even treatment failure [6].

The most current data regarding AMR in zoonotic bacteria are published annually by European Food Safety Authority (EFSA) and European Centre for Disease Control and Prevention (ECDC), but usually with a two-year delay in publishing. According to the recent report on AMR in zoonotic bacteria in 2016 [5], resistance in *Salmonella* and *Campylobacter* is considered of the highest concern. The scope of this review is, therefore, to discuss and emphasise the current trends of AMR in *Salmonella* and *Campylobacter* in the light of the recent EFSA/ECDC report, and the role of whole genome sequencing (WGS) in the surveillance of

For more than a decade, campylobacteriosis has been the most common zoonosis in Europe. Salmonellosis is the second most commonly reported enteric infection, and the leading cause of food-borne outbreaks. *Campylobacter* and *Salmonella* combined accounted for almost 95% of

Salmonellosis is a food-borne gastrointestinal infection caused by zoonotic bacteria *Salmonella* spp. Several thousand serovars of *Salmonella* spp. *enterica* exist, yet only some are causing disease symptoms. Nontyphoidal serovars are transmitted *via* the food chain, whereas typhoidal serovars, the causative agents of typhoid fever, are restricted to humans [8]. Whilst the majority of nontyphoidal *Salmonella* infections are self-limiting and do not require any antibiotic treatment, some cases result in life-threatening systemic infections that must be treated with antimicrobials, primarily fluoroquinolones (FQ) or third-generation cephalosporins [5].

*Campylobacter* spp. are common gut commensals of several animal species, especially birds [9], and the leading cause of gastroenteritis in humans, yet the infections often go unreported. The majority of campylobacterioses are caused by two species, namely *Campylobacter jejuni* and *Campylobacter coli*. Symptoms of campylobacteriosis are also usually mild and self-limiting, although some patients with acute infections that can trigger autoimmune inflammatory conditions need to be treated with antimicrobials, primarily macrolides and FQ [5]. Emergence of

Resistance to these drugs may jeopardise the efficiency of the antimicrobial therapy.

AMR in *Salmonella* and *Campylobacter* along the food production chain.

the reported and confirmed zoonoses cases in 2016 (**Figure 1**).

resistance in *Campylobacter* is common and thus of concern.

**2. Common zoonoses in Europe**

14 Antimicrobial Resistance - A Global Threat

#### **3.1. Prevalence of nontyphoidal** *Salmonella* **in the food chain**

According to the recent report on trends and sources of zoonoses, zoonotic agents and foodborne outbreaks in 2016 published by EFSA/ECDC [7], the declining trend of salmonellosis in Europe has ended. In 2016, there were 94,530 confirmed cases of salmonellosis with the highest notification rate per 100,000 population in Eastern Europe (in average 46.9 per 100,000), mostly on the account of Czech Republic (110) and Slovakia (97.7), followed by Northern (20.6), Western (18.8) and Southern Europe (13.0). Additionally, *Salmonella* was most regularly detected in food-borne outbreaks (22.3%), which have resulted in the highest burden of hospitalisations (45.6% of the total number of hospitalised cases) and deaths (50% of the total number of deaths among outbreak cases) [7]. Outbreaks were linked to several sources, e.g., Polish eggs [14], infant formula [15] and sesame seeds [16].

*Salmonella* was the most prevalent in meat from turkeys (7.74% of the samples tested positive) and from broilers (6.39%), as well as dried seeds (8.0%) [7], which are an important source of infections, especially due to a long shelf life and low moisture [17]. Chicken, turkey and other avian species are commonly inhabited with *Salmonella* without noticeable symptoms [18], which is in addition to the practices in the food production chain [19], considered the highest risk for contamination of meat products. Even though *Salmonella* was significantly less frequently detected in eggs and their products, they remain the most important source of outbreaks [14, 20], most probably due to a large worldwide consumption coupled with low concentrations of *Salmonella* that cannot be detected [7]. Rapid methods with improved sensitivity are thus needed to address this shortfall, e.g., real-time recombinase polymerase amplification [21] or sequence-based methods.

FQ resistance to ciprofloxacin or nalidixic acid that is reflecting similar genetic mechanisms [11] was remarkably high in isolates from poultry meat (**Figure 2**), followed by poultry [5] and has steeply increased since 2004 [27]. That is of concern, because FQ are in addition to thirdgeneration cephalosporins clinically important for the treatment of salmonellosis and several

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Broiler meat (64.7%), broilers (53.8%), turkey (50.5%) and turkey meat (43.7%) were the main sources of FQ resistance. In contrast, isolates from pigs, humans and laying hens had better susceptibility (**Figure 3**) [5, 22]. The ranges of FQ resistance, however, varied extremely among the countries and even regions*.* In Spain, for example, isolates from pigs in Catalonia (50%) [28] and humans in Extremadura (35%) [29] exhibited much higher levels of FQ resis-

FQ resistance in isolates from broilers and meat thereof was in average most prevalent in Southern (62.2, 70.1%, respectively) and Eastern Europe (65.5, 71.5%, respectively) and exceeded 90% in Cyprus, Slovenia and Croatia (**Figure 4**). Of note, only a minority of the MS from Northern Europe reported these data [5]. A rapid increase in FQ resistance is evident in Europe, for instance, in isolates from broilers in Spain resistance increased from 0 [30] to

**Figure 3.** Rates of FQ resistance in isolates of *Salmonella* spp. from various sources*:* broiler meat, broilers, turkeys, turkey

**Figure 4.** Prevalence of FQ-resistant isolates of *Salmonella* spp*.* from broilers, meat thereof and humans in Europe. UK:

**3.3. FQ resistance and third-generation cephalosporins resistance in** *Salmonella*

tance than reported by EFSA/ECDC for Spain (7 and 14.8%, respectively).

other infections, yet both may be used in livestock.

55.6% in just a few years.

United Kingdom [5].

meat, laying hens, humans and pigs, respectively [5, 22]*.*

#### **3.2. Antimicrobial resistance in nontyphoidal** *Salmonella* **spp.**

The most recent data (from 2016) on AMR in *Salmonella* from poultry, meat thereof, and humans are provided by EFSA/ECDC [5], whereas data on other livestock are presented in the last-year report [22]. Generally, as shown in **Figure 2**, the isolates of *Salmonella* spp. along the food production chain tend to be highly resistant to tetracyclines and ciprofloxacin (in average in the EU up to 65%), sulfonamides (56%) and ampicillin (45%) with the highest observed frequency in poultry, meat thereof and pigs, respectively (**Figure 2**). Resistance rates seemed to be higher in Southern or Eastern Europe than in Northern or Western Europe [5, 22]. Such extreme rates of resistance that could indeed reflect an extensive use of these three antimicrobials in livestock [23] are of concern and could be facilitating further dissemination of AMR.

Sulfonamides and ampicillin were the former first-line drugs against salmonellosis [24]. Nevertheless, ampicillin, a critically important antimicrobial [25], is used for treating community acquired pneumonia, complicated severe acute malnutrition and sepsis in neonates and children [26]. In contrast, sulfonamides and tetracyclines are classified as highly important antimicrobials [25]. Sulfonamides are the first-line drugs against urinary tract infections and tetracyclines against *Chlamydia trachomatis* and cholera [26].

**Figure 2.** Prevalence of resistance in *Salmonella* spp*.* AMP: ampicillin, SUL: sulfonamides, CIP: ciprofloxacin, TET: tetracyclines [5, 22].

FQ resistance to ciprofloxacin or nalidixic acid that is reflecting similar genetic mechanisms [11] was remarkably high in isolates from poultry meat (**Figure 2**), followed by poultry [5] and has steeply increased since 2004 [27]. That is of concern, because FQ are in addition to thirdgeneration cephalosporins clinically important for the treatment of salmonellosis and several other infections, yet both may be used in livestock.

#### **3.3. FQ resistance and third-generation cephalosporins resistance in** *Salmonella*

of outbreaks [14, 20], most probably due to a large worldwide consumption coupled with low concentrations of *Salmonella* that cannot be detected [7]. Rapid methods with improved sensitivity are thus needed to address this shortfall, e.g., real-time recombinase polymerase

The most recent data (from 2016) on AMR in *Salmonella* from poultry, meat thereof, and humans are provided by EFSA/ECDC [5], whereas data on other livestock are presented in the last-year report [22]. Generally, as shown in **Figure 2**, the isolates of *Salmonella* spp. along the food production chain tend to be highly resistant to tetracyclines and ciprofloxacin (in average in the EU up to 65%), sulfonamides (56%) and ampicillin (45%) with the highest observed frequency in poultry, meat thereof and pigs, respectively (**Figure 2**). Resistance rates seemed to be higher in Southern or Eastern Europe than in Northern or Western Europe [5, 22]. Such extreme rates of resistance that could indeed reflect an extensive use of these three antimicrobials in livestock [23] are of concern and could be facilitating further dissemi-

Sulfonamides and ampicillin were the former first-line drugs against salmonellosis [24]. Nevertheless, ampicillin, a critically important antimicrobial [25], is used for treating community acquired pneumonia, complicated severe acute malnutrition and sepsis in neonates and children [26]. In contrast, sulfonamides and tetracyclines are classified as highly important antimicrobials [25]. Sulfonamides are the first-line drugs against urinary tract infections and

**Figure 2.** Prevalence of resistance in *Salmonella* spp*.* AMP: ampicillin, SUL: sulfonamides, CIP: ciprofloxacin, TET:

amplification [21] or sequence-based methods.

16 Antimicrobial Resistance - A Global Threat

nation of AMR.

tetracyclines [5, 22].

**3.2. Antimicrobial resistance in nontyphoidal** *Salmonella* **spp.**

tetracyclines against *Chlamydia trachomatis* and cholera [26].

Broiler meat (64.7%), broilers (53.8%), turkey (50.5%) and turkey meat (43.7%) were the main sources of FQ resistance. In contrast, isolates from pigs, humans and laying hens had better susceptibility (**Figure 3**) [5, 22]. The ranges of FQ resistance, however, varied extremely among the countries and even regions*.* In Spain, for example, isolates from pigs in Catalonia (50%) [28] and humans in Extremadura (35%) [29] exhibited much higher levels of FQ resistance than reported by EFSA/ECDC for Spain (7 and 14.8%, respectively).

FQ resistance in isolates from broilers and meat thereof was in average most prevalent in Southern (62.2, 70.1%, respectively) and Eastern Europe (65.5, 71.5%, respectively) and exceeded 90% in Cyprus, Slovenia and Croatia (**Figure 4**). Of note, only a minority of the MS from Northern Europe reported these data [5]. A rapid increase in FQ resistance is evident in Europe, for instance, in isolates from broilers in Spain resistance increased from 0 [30] to 55.6% in just a few years.

**Figure 3.** Rates of FQ resistance in isolates of *Salmonella* spp. from various sources*:* broiler meat, broilers, turkeys, turkey meat, laying hens, humans and pigs, respectively [5, 22]*.*

**Figure 4.** Prevalence of FQ-resistant isolates of *Salmonella* spp*.* from broilers, meat thereof and humans in Europe. UK: United Kingdom [5].

In contrast, the proportions of FQ resistance in humans were significantly lower (11%) [5] and has remained at a relatively stable level since 2009 [31]. The biggest share of FQ resistance was detected in Northern Europe, mostly on the account of Estonia (36.0%), Finland (26.3%), Norway (24.7%) and Ireland (22.9%) (**Figure 4**). Human-associated serovars commonly detected in Europe [32–35] that frequently exhibited FQ resistance were *S.* Infantis (23.4%) and *S.* Kentucky (85.8%) [5].

*S.* Infantis was the most prevalent serovar in broilers and the fourth among human infections. Multi-drug resistance to FQ, sulfonamides and tetracyclines was observed frequently in the isolates from broilers (75.3%), broiler meat (72.6%), as well as in isolates from humans in two MS (Austria and Slovenia) that together with Hungary and Croatia accounted for a majority of the *S.* Infantis isolates from broilers. This indicates the presence of a specific MDR clone prevalent in this geographical region [5]. In addition, resistance to cephalosporins was recorded in the isolates from either humans, food or poultry in Great Britain [48], Switzerland [34], Italy [35], as well as in the USA [49], in some cases located on a plasmid and thus conferring a risk of transfer. Such strains can be transmitted from broilers and broiler meat to

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Whilst *Campylobacter,* with 246,307 confirmed infections in 2016 and 6.1% increase relative to 2015, accounted for the majority of zoonoses in Europe, the death toll was low (0.03%). The highest notification rate per 100,000 population was observed in Eastern Europe (71.4), followed by Western (65.7), Southern (56.3) and Northern Europe (55.0). Czech Republic (228.2)

Campylobacter*s* were most frequently detected in turkeys (65.3%) and meat thereof (11%) as well as in broilers (27.3%) and meat thereof (36.7%) [7], making the poultry food production chain the main source of contamination. This is in concordance to data from several reports [50, 51]. The prevalence in retail poultry meat was, however, reported even up to almost 90% [50]. *Campylobacter* was also detected in cattle [52], pigs [53] and sheep [54]. Contaminated farm environment or equipment as well as the presence of *Campylobacter* in other animals and wildlife, were significantly associated with the prevalence of *Campylobacter* in poultry [55]. Furthermore, recent data suggest that human clinical *C. jejuni* isolates in Central Europe can

be attributed to domesticated poultry, cattle livestock and environmental sources [56].

tion chain, that are oriented towards categorising risks should thus be established [50].

Outbreaks can be traced back to several sources (e.g., raw milk [57], water [58] and chicken liver pate [59]) and even associated with antimicrobial-resistant strains [57]. However, a limited number of highly contaminated products are most probably responsible for the majority of *Campylobacter* infections. Effective and harmonised surveillance systems, especially in the poultry food produc-

*Campylobacter* spp. in 2016 displayed extremely high resistance levels to FQ, which is particularly worrisome, as FQ are used as the first-line drugs against campylobacteriosis. Consequently, in some EU countries, FQ therapy of campylobacteriosis is no longer feasible. In average, the highest share of resistant isolates was detected in poultry and meat thereof, especially in the Member States of Southern and Eastern Europe (**Figure 5**) [5]. Data suggest that the use of FQ in livestock, specifically pigs, selects for FQ-resistant strains and accelerates

humans and may lead to human infections [35].

**4.1. Prevalence of** *Campylobacter* **in the food chain**

**4.2. Antimicrobial resistance in** *Campylobacter*

the dissemination of such strains [60].

and Slovakia (140.5) were the countries with the highest prevalence [7].

**4.** *Campylobacter*

FQ resistance is a result of a complex mechanism, but it is still not fully understood [36]. Many point mutations in the genes encoding for gyrase and topoisomerase, the two enzymes that are inhibited by FQ, were identified as the causative agents [37]. In addition, plasmids may harbour genes for efflux pumps, target protection proteins or drug-modifying enzymes [38].

Resistance to third-generation cephalosporins was rare in humans as well as in livestock [5], yet when combined with FQ resistance, it poses a serious risk to human health, in terms of reducing the efficiency of these drugs against salmonellosis and thus leaving only the reserve antimicrobials as a feasible therapy option [24]. Resistance to cephalosporins is conferred by genes encoding for AmpC β-lactamase as well as for various extended spectrum β-lactamase (ESBL) that can be located on plasmids. Such isolates were observed in Germany [39] and are assumed to have clonally spread from livestock to humans. Worryingly, in Portugal, more than a third of the isolates from broiler meat (39.4%) exhibited resistance to cefotaxime and ceftazidime and in Italy 12% from broilers [5]. In addition, combined resistance to FQ and cephalosporins was detected in poultry and humans in Spain, Belgium and France [40, 41].

*Salmonella* in the food production chain presents an important reservoir of genetic resistance determinants, which could be mobilised and transferred *via* the food chain to either other human pathogens or commensal bacteria [42]. Notably, importation of meat products [43] and travelling in endemic areas [44], where the rates of resistance to critically important antimicrobials are alarmingly high [45], were linked to the global spread of MDR strains.

### **3.4. MDR and combined resistance to fluoroquinolones and third-generation cephalosporins**

In general, 26.5% of the human isolates of *Salmonella* and 50.3% of broiler meat displayed MDR phenotype (defined as resistant to at least three antimicrobials of the nine antimicrobial classes tested). The highest prevalence of MDR isolates from humans was observed in Portugal (51%) and from broiler meat in Slovenia (100%) [5]. MDR strains isolated from pigs in Germany were associated with integrons, which might have an important role in dissemination of resistance [46].

The majority of MDR isolates belonged to serovars *S.* Infantis and *S.* Kentucky. Among human isolates of *S.* Kentucky, which is the seventh most common serovar, MDR was recorded at extremely high levels (76.3%) [5]. *S.* Kentucky ST198 clone that is displaying high-level resistance to ciprofloxacin and frequently also to amoxicillin, streptomycin, spectinomycin, gentamicin, sulfamethoxazole and tetracycline has been imported from North Africa and has been widely spread across Europe in humans and food production chain [32]. In addition, acquisition of extended-spectrum β-lactamase, plasmid-encoded cephalosporinase or carbapenemase in this clone was detected in Mediterranean area [47] and in Poland [33]. Combined resistance was also detected in *S.* Kentucky from humans and livestock in Belgium, Luxembourg, Malta, the Netherlands and Germany [5].

*S.* Infantis was the most prevalent serovar in broilers and the fourth among human infections. Multi-drug resistance to FQ, sulfonamides and tetracyclines was observed frequently in the isolates from broilers (75.3%), broiler meat (72.6%), as well as in isolates from humans in two MS (Austria and Slovenia) that together with Hungary and Croatia accounted for a majority of the *S.* Infantis isolates from broilers. This indicates the presence of a specific MDR clone prevalent in this geographical region [5]. In addition, resistance to cephalosporins was recorded in the isolates from either humans, food or poultry in Great Britain [48], Switzerland [34], Italy [35], as well as in the USA [49], in some cases located on a plasmid and thus conferring a risk of transfer. Such strains can be transmitted from broilers and broiler meat to humans and may lead to human infections [35].
